Difference between revisions of "Part:BBa K3893015"

Latest revision as of 04:32, 18 December 2021

Down part of cassette for dsRNA constitutive production 4

Ppart of the Modular Platform for dsRNA production in the proyect AgroBactory 593.

Lower part of the cassette for constitutive dsRNA production, which is composed of a GFP transcription unit that functions as a dropout (BBa_K3893018), Shine Dalgarno (BBa_K3893021), RBS (BBa_K3893022), promoter T7 (BBa_K3893020) and terminator (BBa_K3893006).

Assembly system

Before constructing the modified plasmids and using them, we took into account the following:

• Have a Biobrick-compatible backbone, in case you don't have it you could modify it with PCR overhang and add RFC10 prefix and suffix.
• The following restriction enzymes are used: EcoRI, PstI, SpeI, XbaI, BglII and KpnI. Therefore the dsRNA sequence should not contain these restriction sites.
• Previously carry out PCR overhang to the dsRNA sequence to add the restriction sites: BglII and KpnI.

Once the above considerations have been met, the following assemblies are performed:

1. We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI.

Step 1

2. Assembly 3A adapted: We digested the UP plasmid with EcoRI - SpeI, the DP plasmid with XbaI - PstI and a Biobrick compatible backbone with EcoRI - PstI. Then we ligated and obtained as a product the modified plasmid (dsRNA receptor).

Step 2

3. We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.

Step 3

Sequence and Features